Beneficial Uses of Viruses in Biotechnology

1. Beneficial Uses of Viruses in Biotechnology • Gene therapy • Vaccines and vaccine carrier / delivery vehicles • Antibacterial agents • Basic knowledge of cell metabolic processes • Vectors for mammalian, plant and insect cell protein expression systems • Peptide display – industrial /pharmaceutical / medical reagent development

2. Gene therapy: Viruses as gene delivery vehicles • Necessary viral properties • Use a virus that persists in humans, and shows moderate level of long-term gene expression • Need to clone in foreign DNA (where, how much) – develop cloning sites, need large cloning capacity • Virus must target specific cell-type – cell-type-specific receptor mediated attachment and uptake – can genetically engineer for receptor

3. Gene therapy: Viruses as gene delivery vehicles • Manufacture • propagate virus in specific mammalian cell line – expensive • Biosafety - mutate to produce attenuated virus (low virulence) or crippled virus (reduced pathogenicity) - but generally low titre • To circumvent low titre – Helper virus or transgenic cell line provides packaging / replication functions in trans for non-replicating delivery virus • viral packaging - protects and stabilises DNA from degradation

4. Gene therapy: Viruses as gene delivery vehicles • Host response • Do not want a strong immune response to viral vector (& its associated payload) – leads to rapid clearance of delivery virus • Thus, use viruses with • rare serotypes • low seroprevalence • low-level replication or non-replicating virus • E.g. lentiviruses

5. Viruses: Direct use in vaccines • Against autologous virus • attenuated or inactivated virus • viral subunits - usually structural proteins, genetic vaccines • cross-reactivity - study relatedness of virus strains • Against heterologous virus • Viral structural proteins or “virus-like particles” (VLPs) can be made to carry heterologous pathogen epitopes • Must produce proteins, VLPs in quantity to high purity • e.g. rabies virus glycoprotein, HPV

6. Viruses as DNA vaccine delivery vehicles • Preferred Immunological Properties • Want weak immune response (IR) to viral delivery vehicle • Weak / moderate IR to delivery vehicle results in enhanced response to DNA payload • Strong IR to delivery vehicle may provoke toxic over-response, clear the vaccine too rapidly for a response to develop to the payload, or can swamp response to payload • Therefore: • Use viruses having rare serotypes (low seroprevalence) • low virulence or non-pathogenic viruses

7. Viruses as DNA vaccine delivery vehicles • Develop a viral delivery vehicle • study gene function, engineer suitable cloning sites • amount of DNA vaccine that can be cloned and packaged is limited by capsid size / viral packaging mechanism • Cell targeting – DNA delivery • viral engulfment by antigen presenting cells • cell-specific receptor-mediated uptake • Manufacture • Prefer a virus that replicates to high titre • Prefer a virus with a long survival half life outside host cell • Viral packaging of DNA vaccine protects and stabilises DNA from degradation • Need suitable production-host cells – if using attenuated virus (eg vaccinia vectors) - may need to provide some packaging / replication functions in trans

8. Plasmid DNA makes encoded HIV protein in cells of the body Vector Vaccines for HIV-1 Virus-like particle with outer surface display of epitopes Epitope Display Vectors Live AttenuatedViral Vectors Adenovirus Modified Vaccinia (MVA) Replicon Vaccines: Virally encapsidated plasmid vaccine DNA from HIV is Cloned into Various Vectors

9. Plasmid DNA makes encoded HIV protein in cells of the body Vector Vaccines for HIV-1 DNA from HIV is Cloned into Various Vectors

10. Viral genetic elements used to construct Eukaryotic expression plasmid vectors • Viruses are highly efficient replicators & viral gene expression is adapted to eukaryotic systems • very strong promoters (CMV immediate / early promoter) • small introns (CMV intron) • regulatory elements often constitutive - require only host factor binding (porcine circovirus (PCV) capsid promoter / enhancer) • Therefore mine regulatory elements from viruses • Promoters, enhancers, polyadenylation signals, introns, replication origins, IRES elements.

11. Vector Vaccines for HIV-1 Virus-like particle with outer surface display of epitopes Epitope Display Vectors DNA from HIV is Cloned into Various Vectors

12. Use of insect Baculovirus : Autographa californica nuclear polyhedrosis virus (AcNPV): Foreign gene (HIV-1 gag) inserted under Baculovirus strong late promoter, polh -transient production of HIV Virus-Like Particles in cultured insect cells A. Meyers, E.P. Rybicki.

13. Tobacco mosaic virus Viruses for Peptide display: M13 Phage or plant virus (TMV) Coat Protein Fusions • Need : • non-enveloped virus • many repeat capsid subunits • ordered capsid array - amplified display • external loops or termini available for peptide addition via gene fusion Mass peptide display on outer surface of TMV particle N C 60S loop Assembly of mixed TMV capsids carrying epitope variants = useful vaccine vs highly variable pathogen TMV VIRION

14. Vector Vaccines for HIV-1 Live AttenuatedViral Vectors Adenovirus Modified Vaccinia (MVA) DNA from HIV is Cloned into Various Vectors

15. Live Attenuated Viral Vectors at UCT Modified Vaccinia Ankara (MVA) • HIV-1 vaccine development at UCT • Recombinant MVA (rMVA) expressing HIV-1C gag and env genes • Used in a Prime-Boost immunisation regimen • prime immune response with plasmid vaccine expressing gag and env • boost to broaden / increase response with rMVA expressing gag and env DNA prime rMVA boost

16. Vector Vaccines for HIV-1 Replicon Vaccines: Virally encapsidated plasmid vaccine DNA from HIV is Cloned into Various Vectors

17. Replicon Vaccines: Virally encapsidated plasmid vaccine • Adenovirus 5, Adeno-associated virus • Bacteriophage vectors e.g. Lambda or M13 • clone foreign DNA into Lambda genome • Large cloning capacity • passive uptake by immune cells and complement mediated uptake • Non-pathogenic for humans - safe • Highly stable vehicle - can dehydrate • Cheap to make – high titre production in E. coli

18. Macrophage Dendritic cell Cell transcribes DNA. Vaccine protein is expressed on cell surface Principle: Mammalian expression control elements l DNA l DNA Antigen gene Phage broken down. Vaccine-encoding DNA released Vaccine expression cassette cloned into bacteriophage l DNA Immune response Grow l phage in E. coli & purify Antigen – presenting cells engulf l particles Inoculate - injection / oral

19. Bacteriophage: viral antibacterial agents • Advantages: • Useful where multiple antibiotic resistance has developed • host specific - won't kill off commensal bacteria • Rapid action – exponential replication • self-limiting infection once pathogenic bacteria are killed • cheap - single dose - self propagates • Disadvantage - strain specific • need to generate, keep and archive large bank of phage serotypes • need accurate diagnosis • must give cocktail of phage types to prevent bacterial escape • Also use for detecting pathogenic bacteria - phage infects bacterial lawn - assay plaques by antibody or by phage-encoded marker gene expression Multi drug resistant Pseudomonas